Figure 4. The GRA is under the control of the P. aeruginosa LASI/RHLI and IQS QS systems.

L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) is the main GRA released by P. aeruginosa. (A) Histograms show seed germination percentage 4 days upon sowing seeds in absence (MS) or presence of WT P. aeruginosa (PAO1) or mutant P. aeruginosa strains as indicated. PAO1 is the reference WT P. aeruginosa strain from which all the QS mutant strains were derived. Seeds and P. aeruginosa cells were separated by 2 cm. Data represent mean ± standard deviation (four replicates, n = 150–200). Statistical treatment and lower case letters as in Figure 1B. (B) Same experiment as in A. using P. aeruginosa ΔambA-E mutants, as indicated, each affected in individual AMB operon genes. Data represent mean ± standard deviation (three replicates, n = 100–150). Statistical treatment and lower case letters as in Figure 1B. (C) AMB represses Arabidopsis seed germination in a DELLA-dependent manner. Pictures show representative Arabidopsis plants 4 days after sowing WT and Δdella seeds in presence of different AMB concentrations as indicated. The graph shows quantification of the germination percentage of WT and Δdella seeds exposed to AMB. The red dashed line indicates the concentration of synthetic AMB (50 µM) having the same GRA as that present in germination plates containing 0.8 mg/ml of PAO1 extracts (see Figure 4E). Data represent mean ± standard deviation (three replicates, n = 150–200). (D) AMB represses Arabidopsis seed germination in an ABA-dependent manner. Histograms show germination percentage of WT, aba1, abi3 and Δdella 3 days after sowing seeds in absence (MS) or presence of 50 µM of synthetic AMB. Data represent mean ± standard deviation (two replicates, n = 100–150). Statistical treatment and lower case letters as in Figure 1B. (E) Synthetic AMB introduces a GRA in ΔambE extracts equivalent to that of PAO1 extracts. ΔambE extracts, which lack AMB, were supplemented with synthetic AMB so as to obtain the same amount of AMB naturally present in PAO1 extracts. Pictures show representative Arabidopsis plants 4 days after sowing WT seeds in absence (MS) or presence of WT P. aeruginosa extracts (PAO1extract) or ΔambE P. aeruginosa extracts (ΔambEextract) or AMB-supplemented ΔambE extracts (ΔambEextract + AMB). 0.8 mg/ml of WT P. aeruginosa extract was used. The graph shows quantification of the germination percentage of WT seeds exposed to the different extract concentrations as indicated. Note that the supplemented ΔambE extract (ΔambEextract + AMB) has a GRA equivalent, if not higher, than that of PAO1 extract. The red dashed line indicates the concentration of PAO1 extract (0.8 mg/ml) having the same GRA as that present in germination plates containing 50 µM synthetic AMB (see Figure 4C). Data represent mean ± standard deviation (six replicates, n = 250–300).

Figure 4—figure supplement 1. The GRA is under the control of the P. aeruginosa LASI/RHLI and IQS QS systems.

(A) Pictures show Arabidopsis plants 4 days after sowing WT seeds in germination medium containing a given Pseudomonas species as indicated. PAO1 is the reference WT P. aeruginosa strain from which the QS mutant (ΔpqsA and ΔambE) and AMB operon overexpressing (AMBox) strains were derived. (B) Same experiment as in A. Heatmap representation mean of the germination percentage of seeds according to time after sowing and distance from bacteria (four replicates, n = 150–200). Seeds were sowed in germination plates in absence (MS) or presence of P. aeruginosa strains as indicated in blue. PAO1 is the reference WT P. aeruginosa strain from which the QS mutant (ΔpqsA, ΔpqsH, ΔambE, ΔlasIΔrhlI) and AMB operon overexpressing (AMBox) strains were derived.

Figure 4—figure supplement 2. Approaches used in this study to identify compounds released by P. aeruginosa with a germination repressive activity (GRA).

Figure 4—figure supplement 3. Unsupervised and OPLS-DA results from metabolomic analysis 1.

(A) PCA analysis using HRMS² data from four different extracts: PAO1, ΔpqsA, ΔambE and ∆lasI∆rhlI (two replicates). (B) Scores and model overview of the OPLS-DA realized between strains releasing a GRA (PAO1 and ∆pqsA), classified as a group named ‘Active’ (blue color), or not releasing a GRA (∆ambE and ∆lasI∆rhlI), classified as a group named ‘Not Active’ (red color). All features are reported in Supplementary file 3, sheet ‘MS-MS analysis 1’.

Figure 4—figure supplement 4. Unsupervised and OPLS-DA results from metabolomic analysis 2.

(A) PCA analysis using HRMS² data from three different extracts: PAO1, AMBox and ΔambE (three replicates). (B) Scores and model overview of the OPLS-DA realized between strains releasing a GRA (AMBox), classed as ‘Active’ (blue color) or not releasing a GRA (∆ambE), classed as ‘Not Active’ (red color). All features are reported in Supplementary file 3, sheet ‘MS-MS analysis 2’.

Figure 4—figure supplement 5. m/z 327.12 is the Mass Spectrometry (MS) signature of AMB.

(A) Specific cluster of the molecular network realized on UHPLC-HRMS² data acquired on extracts from strains releasing (PAO1 and ∆pqsA) or not releasing a GRA (∆ambE and ∆lasI∆rhlI). Color mapping grouped the MS/MS spectrum as following: blue if acquired from WT (PAO1) or ∆pqsA (∆pqsA) P. aeruginosa extracts, red if acquired from ∆ambE (∆ambE) or ∆lasI∆rhlI (∆lasI∆rhlI) mutant P. aeruginosa extracts and grey if acquired from analytical blanck (H₂O) or culture media (MS) extracts. (B) Peak intensities visualization plot of the extracted ion trace from feature m/z 327.12 at 0.46 min. This plot shows the intensity of this particular feature across samples of AMBox [n = 3], analytical blanck [n = 2], ∆ambE [n = 3], culture media extract [n = 3] and PAO1 [n = 3], in this respective order.

Figure 4—figure supplement 6. Isolation of the GRA released by P.aeruginosa.

(A) Semi-prep HPLC-UV (scan 210–600 nm) chromatogram of the fractionation of the active PAO1 extract. Fraction F1 presented strong inhibitory seed germination activity. (B) Semi-prep HPLC-ELSD chromatogram of the isolation of the active principle from the AMBox extract localized in fraction 37 (F37). The structure of the AMB was determined by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry HRMS.

Figure 4—figure supplement 7. Fraction F37 contains AMB and a GRA.

Fraction F37 was solubilized in water and its AMB content (referred as AMBi) was measured by NMR, which allowed to prepare a stock solution containing 10 mM AMBi. Dose response of WT (Col-0) and Δdella seeds treated with different concentrations of AMBi as indicated. Data represent mean ± standard deviation (2 to 5 replicates, n = 150–300).

Figure 4—figure supplement 8. Quantification of AMB in Pseudomonas extracts.

(A) Calibration curve using synthetic AMB used for quantification. Data represent mean ± standard deviation (three replicates). (B) Quantification of AMB in Pseudomonas extracts at 1 mg/mL. Data represent mean ±standard deviation (three replicates). Bioactivity of the same extracts were represented in Figure 4E.

Figure 4—figure supplement 9. ∆ambE extracts supplemented with AMBi or synthetic AMB contain the same GRA.

1 mg/ml of PAO1 extract (containing 20 μM of AMB) strongly inhibits germination of WT (Col-0) seeds (grey boxplot) but not that of Δdella mutant seeds (blue boxplot). 1 mg/ml of ΔambE extract does not inhibit germination. An ΔambE extract supplemented with 20 μM of synthetic AMB (ΔambE + AMB) or AMB isolated from P. aeruginosa (ΔambE + AMBi) has the same GRA as an extract isolated from P. aeruginosa (PAO1). Statistically significant differences were assessed by one-way ANOVA followed by a Tukey HSD test (p<0.05, six replicates for each conditions, n = 250–300).

Figure 4—figure supplement 10. The oxyvinylglycine AVG does not inhibit Arabidopsis seed germination.

Histograms show germination percentage of Arabidopsis seeds sown in absence or presence of different concentrations of AVG, as indicated. Data represent mean ± standard deviation (three replicates, n = 150–200) and statistically significant differences were assessed by one-way ANOVA followed by a Tukey HSD test (p<0.05).

Figure 4—figure supplement 11. Presence of the ambABCDE operon in at least 15 other P. aeruginosa strains.

Non-exhaustive list of 15 different P. aeruginosa strains containing ambABCDE genes in there genomes. Ortholog genes and structural operon organization where defined based on Pseudomonas database (http://www.pseudomonas.com/).